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Biomedical subjects

S D Holmes

Publications and source records attributed to S D Holmes.

At least 19 recordsLinked to original sources
PubMed2000

Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor.

The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8(+) than CD4(+) T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.

CD4-Positive T-Lymphocytes↗
PubMed2000

Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase: a potential new risk factor for coronary artery disease.

A specific and robust immunoassay for the lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), platelet-activating factor acetylhydrolase, is described for the first time. The immunoassay was used to evaluate possible links between plasma Lp-PLA(2) levels and atherosclerosis risk amongst susceptible individuals. Such an investigation was important because Lp-PLA(2) participates in the oxidative modification of low density lipoprotein by cleaving oxidised phosphatidylcholines, generating lysophosphatidylcholine and oxidised free fatty acids. The majority of Lp-PLA(2) was found associated with LDL (approximately 80%) and, as expected, enzyme levels were significantly positively correlated to LDL cholesterol. Plasma Lp-PLA(2) levels were significantly elevated in patients with angiographically proven coronary artery disease (CAD) when compared with age-matched controls, even though LDL cholesterol levels did not differ significantly. Indeed, when included in a general linear model with LDL cholesterol and other risk factors, Lp-PLA(2) appeared to be an independent predictor of disease status. We propose, therefore, that plasma Lp-PLA(2) mass should be viewed as a potential novel risk factor for CAD that provides information related to but additional to traditional lipoprotein measurements.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2000

Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils.

BACKGROUND: Eosinophils, basophils, and mast cells are believed to be the central tenet cells in allergic conditions including allergic rhinitis, asthma, and eczema. The molecular mechanisms underlying the recruitment of these cells to sites of allergic inflammation are poorly understood. OBJECTIVES: Our aim was to identify a common adhesion molecule that could potentially be responsible for mediating the recruitment of the allergic cell types to the lungs and other sites of allergy. METHODS: We have cloned a sialoadhesin molecule from a human eosinophil library with the use of expressed sequence tag technology and characterized its expression on allergic cells by the use of flow cytometry and specific mAbs. RESULTS: With the use of expressed sequence tag sequencing, we have identified a novel siglec molecule, SAF-2. SAF-2 has homology with other sialoadhesin family members (CD33 and siglec-5) and belongs to a subgroup of the Ig superfamily. SAF-2 is a 431-amino acid protein composed of 3 Ig domains with a 358-amino acid extracellular domain and a 47-amino acid tail. SAF-2 is highly restricted to eosinophils, basophils, and mast cells. Antibodies to SAF-2 do not modulate Ca(++) mobilization or chemotaxis of human eosinophils induced by eotaxin. CONCLUSION: SAF-2 is a highly restricted sialoadhesin molecule, which may be useful in the detection and/or modulation of allergic cells.

Antigens, CD↗
PubMed1999

Reactivity and assay restriction profiles of monoclonal and polyclonal antibodies to acid phosphatases: a preliminary study.

The development of secure diagnostic immunoassays requires, among others, rigorous characterisation of potential antibody reagents. The reactivity profiles of seven antibodies (six monoclonal [MAb] and one polyclonal [PAb]) with putative specificity for tartrate-resistant acid phosphatase (TRAP) and/or osteoclasts were evaluated in enzyme-linked immunosorbent assay (ELISA) and/or immunocytochemistry. MAbs 2H1, 4E6 and 5Cl demonstrated assay restriction: exhibiting reactivity only in ELISA. The remaining three MAbs (G211D, G312G and V35B) and the PAb 8023 recognised recombinant TRAP (rTRAP) in ELISA and native acid phosphatases in selected tissues and cell lines. The latter were cytochemically assessed for both tartrate-sensitive acid phosphatase (TSAP) and TRAP. V35B showed reactivity against the monocytic leukaemia cell line U937 and guinea pig kidney tissue (both TSAP+ and TRAP+) and ECV304 (TSAP+) cells. Interestingly, the reactivity of MAb G211D co-localised with TRAP activity in the membrane of osteoclasts but also detected cytoplasmic components in U937 cells and human embryonic lung fibroblasts (TRAP+ and TRAP+). G211D exhibited immunoreactivity against placental trophoblasts (positive for total AP). Intriguingly, MAbs 2H1, 4E6, 5Cl and PAb 8023 cross-reacted with potato acid phosphatase in ELISA, suggesting reactivity to conformationally similar epitopes. Thus, some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5Cl and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.

Acid Phosphatase↗
PubMed1999

Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14.

Urotensin-II (U-II) is a vasoactive 'somatostatin-like' cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

Amino Acid Sequence↗
PubMed1999

Anti-human RP105 sera induces lymphocyte proliferation.

Cellular environment dictates whether antigen binding to the B lymphocyte receptor together with co-stimulatory molecules will result in proliferation, anergy, or apoptosis. Murine RP105 is a member of the leucine-rich repeat family of proteins, which is specifically expressed on mature B cells. Monoclonal antibodies to the murine RP105 induce proliferation and protect B cells from apoptosis, suggesting an important regulatory role in murine B lymphocyte function. We identified a human RP105 homolog and mapped the gene to chromosome 5q12.3-13.1. Tissue distribution analysis shows that the transcript is found predominately in lymphoid tissues including spleen, tonsils, appendix, and peripheral blood leukocytes. Polymerase chain reaction analysis of isolated primary human cell populations confirms that mRNA exists in spleen B lymphocytes and monocytes but not T lymphocytes. Western blot analysis demonstrates specific expression of human RP105 in human B lymphocytes. Murine anti-human RP105 sera was generated using DNA immunization. The antisera contained antibodies that recognized and bound to human B lymphocytes from both spleen and peripheral blood as assessed by flow cytometry. Assessment of biological function showed that human peripheral blood leukocytes incubated with anti-RP105 sera were induced to proliferate as measured by tritiated thymidine incorporation. Moreover, anti-CD40 and interleukin-4-treated cells but not those exposed to anti-RP105 sera produced soluble CD23, suggesting distinct functional roles. This is the first demonstration of both the existence of RP105 protein on human B lymphocytes and its role in the regulation of B lymphocyte activation.

Animals↗
PubMed1999

The tissue distribution of the human beta3-adrenoceptor studied using a monoclonal antibody: direct evidence of the beta3-adrenoceptor in human adipose tissue, atrium and skeletal muscle.

OBJECTIVE: To develop a monoclonal antibody that recognises an epitope of the native beta3-adrenoceptor expressed on the extracellular surface of human cells and tissues. DESIGN: A high affinity monoclonal antibody, Mab72c, was raised against the human beta3-adrenoceptor expressed on a transfected mammalian cell line. RESULTS: In CHO (Chinese hamster ovary) cells transfected with beta3-adrenoceptor cDNA, antibody labelling was found to be proportional to receptor density measured by the binding of the radiolabelled beta-adrenoceptor antagonist, [125I]-iodocyanopindolol. The use of Mab 72c has demonstrated the expression of the beta3-adrenoceptor in a variety of human tissues, including gall bladder, prostate and colon, where a mRNA signal had been detected previously. This study also provides the first direct demonstration of the expression of beta3-adrenoceptors in human skeletal muscle, atrium and adipose tissue. CONCLUSION: The development of this antibody represents an important addition to the armentarium of reagents that are available to study the localisation of beta3-adrenoceptors in human tissues.

Adipose Tissue↗
PubMed1999

Cloning and expression of single chain antibody fragments in Escherichia coli and Pichia pastoris.

The potential of recombinant antibody fragments is likely to be fulfilled only if they can be produced routinely at high concentrations. We have compared the ability of Escherichia coli and Pichia pastoris to produce functional recombinant single chain antibody (scAb) fragments. Two scAb fragments were expressed, an antihuman type V acid phosphatase (TRAP) and an anti-Pseudomonas aeruginosa lipoprotein I. We report here that, while expression from P. pastoris resulted in a significantly increased level of expression of the anti-TRAP scAb compared to E. coli, neither fragment was able to bind its target antigen as well as the bacterial product.

Acid Phosphatase↗
PubMed1999

Changes in calcium homoeostasis in patients undergoing liver transplantation: effects of a single infusion of pamidronate administered pre-operatively.

Bone turnover, bone loss and fracture risk increase after liver transplantation. It has been postulated that peri-operative administration of a bisphosphonate might prevent bone loss and reduce fracture rate. We studied the effects of a single pre-operative dose of pamidronate on biochemical parameters of skeletal metabolism in the first month after liver transplantation. In a randomized, single-blind study, six of 12 patients with chronic liver disease received 60 mg of pamidronate intravenously on a single occasion 1-30 days before transplantation. Six other patients undergoing transplantation received no pamidronate. We measured serum calcium, phosphate, albumin, bone-specific alkaline phosphatase, plasma parathyroid hormone and tartrate-resistant acid phosphatase before pamidronate infusion and at frequent intervals during the first 30 post-operative days. In treated patients, plasma parathyroid hormone increased 12-fold over baseline values and remained elevated in comparison with baseline at days 26-30; serum calcium and phosphate fell significantly, returning to normal at around day 14 post-operatively. There were no significant changes in any parameter in the untreated group. No changes in bone formation or resorption markers were observed in either group. The large increase in plasma parathyroid hormone concentrations in the treated group is probably secondary to the fall in serum calcium. The magnitude of the increase is much greater than that seen after pamidronate infusion in other patient groups. The lack of change in, or correlation of, serum calcium and plasma parathyroid hormone in the untreated group suggests that additional factors release calcium from bone after liver transplantation, presumably by increasing bone resorption.

Adult↗
PubMed1997

Effects of intracerebroventricular infusion of leptin in obese Zucker rats.

The obese Zucker rat (OZR) exhibits a missense mutation in the cDNA for the leptin receptor, producing a single amino acid substitution in the extracellular domain of the receptor. A mutation in the leptin receptor gene of the db/db mouse prevents the synthesis of the long splice variant of the receptor. The possibility that the OZR, like the db/db mouse, is refractory to the actions of murine leptin was tested by infusing the protein intracerebroventricularly via a minipump for 7 days. Lean Zucker rats (LZR) infused with leptin acted as positive controls, and other groups of OZR and LZR were infused with vehicle. In LZR, leptin reduced bodyweight and food intake and increased brown adipose tissue (BAT) temperature. Plasma corticosterone increased (61%) in these rats, and plasma triglycerides fell (78%). Leptin treatment improved tolerance to an oral glucose load (16% reduction in the area under the blood glucose curve) while lowering plasma insulin. In OZR, the actions of leptin were blunted. Food intake was slightly, but not significantly, reduced. Although there was a reduction in the rate of increase in body mass, the effect of leptin was about half that seen in LZR. BAT temperature and glucose tolerance were unchanged. In contrast to the elevated plasma corticosterone seen in LZR, leptin reduced the level of this hormone (27%) in OZR. In OZR and LZR treated with leptin, the plasma leptin levels were increased 24-fold and 47-fold, respectively. The results suggest that leptin retains some efficacy in OZR, although these rats are less responsive than LZR.

Adipose Tissue, Brown↗
PubMed1996

The thiazolidinedione insulin sensitiser, BRL 49653, increases the expression of PPAR-gamma and aP2 in adipose tissue of high-fat-fed rats.

The effects of the thiazolidinedione insulin sensitiser BRL 49653 on plasma leptin concentrations and on epididymal fat OB, PPAR-gamma and aP2 mRNA expression were examined in high-fat-fed and high-carbohydrate-fed adult Wistar rats. Diets were given for 4 weeks, with BRL 49653 (10 micromol/kg/day) administered by oral gavage for the last 4 days. Treatment with BRL 49653 reduced plasma leptin concentrations in high-fat-fed rats from 2.34 +/- 0.19 (n=9) to 1.42 +/- 0.09 (n=9) ng/ml (p<0.001). Plasma leptin was unaffected by BRL 49653 in the high-carbohydrate-fed rats. There was no difference in OB mRNA expression between high-fat-fed and high-carbohydrate-fed rats, with or without treatment. PPAR-gamma and aP2 mRNA expression were significantly increased in the high-fat-fed rats treated with BRL 49653 (p < 0.01 and p < 0.001 respectively), but not in carbohydrate-fed rats.

Adipose Tissue↗
PubMed1995

Neutralizing murine monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library.

Conventional hybridomas and combinatorial Ab libraries were used to develop neutralizing murine mAbs to human IL-5. Mice were immunized with rIL-5. Spleens from two mice were used to generate hybridomas. Spleens from an additional three mice were used to construct a combinatorial library. In both instances, Abs were identified and selected by ELISA using 96-well plates coated with rIL-5. These Abs were tested for the ability to block binding of iodinated rIL-5 to the alpha-chain of the human IL-5 receptor (IL-5R alpha) and to inhibit proliferation of IL-5-dependent cells. By hybridoma technology, 16 mAbs were obtained, 11 of which blocked binding to IL-5R alpha, including three that inhibited proliferation. Quantitative binding assays and sequence analysis revealed that these latter three mAbs were closely related. Combinatorial cloning and selection by phage display was used to isolate 24 bacterial colonies secreting Fabs that bound to 125I-rIL-5 and to rIL-5-coated plates. Sequencing of 10 of the Fabs indicated that four unique Abs were obtained, comprising one predominant VH paired with one of two different VL. The sequence of the Fabs was distinct from the sequences of the neutralizing mAbs. In contrast to the mAbs, none of the Fabs blocked binding of 125I-IL-5 to IL-5R alpha or neutralized the biologic activity of IL-5. The inability to identify neutralizing Fabs was shown not to result from their monovalency, because a Fab derived from one of the neutralizing mAbs, by cloning and expression of its Fd and kappa light chains, retained neutralizing activity. By chain shuffling, pairing of the Fd fragment of the heavy chain of one of the neutralizing mAbs (2B6), with the light chain library derived from the IL-5-immunized mice, neutralizing Fabs were obtained. These Fabs contained light chain sequences closely related to the original light chain of 2B6. Hence, chain shuffling allowed detection of a light chain sequence that was not evident upon two-chain combinatorial selection. The results reveal differences in the Abs obtained from a combinatorial library vs hybridomas and demonstrate how these approaches can be used in concert to select mAbs with neutralizing activity.

Amino Acid Sequence↗
PubMed1995

Generation and characterization of monoclonal antibodies to human type-5 tartrate-resistant acid phosphatase: development of a specific immunoassay of the isoenzyme in serum.

We have characterized four monoclonal antibodies (mAbs) to the purple ("tartrate-resistant," band 5) acid phosphatase of the human osteoclast (TRAP) and used these to develop a specific serum immunoassay. All four mAbs are of high affinity (Ka = 1-5 x 10(8) L/mol) with a very fast Kassoc (0.2-2.0 x 10(5) L mol-1 s-1) and a moderate Kdissoc (1-3 x 10(-3) s). Two of the mAbs were selected to develop a time-resolved fluorescence immunoassay to measure serum concentrations of TRAP. The mean serum immunoreactive TRAP in a group of healthy premenopausal women and men was 3.7 +/- 1.8 micrograms/L (mean +/- SD) and 3.5 +/- 1.6 micrograms/L, respectively. Significantly higher concentrations of TRAP were found in postmenopausal women (6.3 +/- 2.3 micrograms/L) and in eight patients with Gaucher disease (19.3 +/- 4.7 micrograms/L). Further studies are required to investigate the value of serum TRAP as a marker of bone resorption.

Acid Phosphatase↗
PubMed1988

Diamphenethide--a reassessment of its pharmacological action.

At a concentration of 1 x 10(-4) M (28.84 micrograms/ml), with a solvent concentration of 1.0% (v/v) ethanol, the deacetylated (amine) metabolite of diamphenethide (DAMD) causes an initial stimulation of activity, followed by suppression, leading to a paralysis within 3 h. These changes are accompanied by an increase in muscle tone of more than 200 mg. However, ethanol alone at a concentration of 1.0% (v/v) causes an initial stimulation of activity and increase in muscle tone (approximately 550 mg). If the concentration of DAMD is kept at 1 x 10(-4) M (28.84 micrograms/ml) but the solvent concentration reduced [e.g., 0.05% (v/v) dimethyl sulphoxide], then only a suppression of motility and flaccid paralysis are observed. This response is also seen at the lower concentration of 10 micrograms/ml, which corresponds to the maximum blood levels of DAMD in vivo. The sodium ionophore monensin induces a suppression of motility, leading to a rapid flaccid paralysis (in approximately 1.5 h at 1 x 10(-7) M, and within a few minutes at higher concentrations). Ouabain, an inhibitor of Na+/K+-ATPase activity, also causes a suppression of motility, but this is accompanied by an increase in muscle tone, leading to a spastic paralysis (in approximately 2.5 h at 1 x 10(-3) M, and 3.5 h at 1 x 10(-4) M). Pretreatment with ouabain (1 x 10(-3) M for 15 min) followed by monensin (1 x 10(-5) M) reverses the original effect of monensin by inducing a rapid spastic paralysis (in approximately 50 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetanilides↗
PubMed1987

Ovarian epidermal growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement.

Numerous data indicate that epidermal growth factor has important effects on cultured granulosa cells. However, most of the few attempts to detect epidermal growth factor in ovarian tissue have been unrevealing. In this study, ovarian epidermal growth factor-like activity was easily detected by a radioreceptor assay based on the A431 cell line but not by an immunoassay for mouse epidermal growth factor. The concentration of this activity in follicular fluid from small porcine ovarian follicles was higher than that in fluid from medium or large follicles or serum (p less than 0.01), but lower than that in salivary gland extracts. Receptor-active epidermal growth factor-like peptides could function as local ovarian regulators.

Animals↗
PubMed1986

Rat Sertoli cells secrete a growth factor that blocks epidermal growth factor (EGF) binding to its receptor.

The conditioned medium from Sertoli cells contains a potent mitogen(s) that can markedly stimulate the proliferation of 4 different cell lines of endoderm or mesoderm origin in the presence or absence of serum. With A431 cells, conditioned medium produced in a dose-dependent manner up to a 5.2-fold increase in cell number after 5 days in culture. Addition of follicle-stimulating hormone (FSH), testosterone, retinol, and insulin to the Sertoli cells increased the secretion of the mitogenic activity. The ability of Sertoli cell conditioned medium (SCCM) to displace 125I-labeled epidermal growth factor (125I-EGF) from formalin-fixed A431 cells was also examined. The SCCM from Sertoli cells incubated with insulin contained 1.42 ng eq of EGF/ml; testosterone, retinol, and FSH (in the presence of insulin) further increased the secretion of this EGF competing activity to 2.09, 2.56, and 3.22 ng eq/ml, respectively. The amount of EGF competing activity was positively correlated with mitogenic activity. Separation of SCCM by gel filtration on Bio-Gel P-10 produced three major peaks of EGF-competing activity at apparent Mr = 1800-2100, 3800-4200, and 8000-9500. Chromatographing SCCM (in the presence of protease inhibitors) on size exclusion high performance liquid chromatography revealed two peaks of EGF competing activity at Mr about 8000 and 2000 coincident with and proportional to peaks of mitogenic activity. This activity was heat-sensitive and resistant to reducing agents, and addition of an equivalent amount of EGF as that present in SCCM produced an inhibition in growth of the A431 cells compared to a 3-fold stimulation with SCCM. Thus, the Sertoli cells secrete a potent mitogen that is distinct from EGF and alpha TGF. This factor that we have termed Sertoli cell-secreted growth factor is hormonally regulated by FSH, testosterone, and retinol and may play an important role in controlling spermatogenesis.

Animals↗
PubMed1986

Delta-9-tetrahydrocannabinol stimulates ABP secretion from rat Sertoli cells in vitro.

The effect of delta-9-tetrahydrocannabinol (THC) on rat Sertoli cell function was investigated. THC significantly increased ABP secretion by 1.5- to 2.1-fold but did not consistently enhance the stimulation of ABP induced by FSH, testosterone or dibutyryl cyclic AMP. ABP was measured by steady-state polyacrylamide gel electrophoresis, DEAE Bio-Gel and immunoassay; all three methods gave similar results. The minimal concentration of THC that stimulated ABP was 10 ng/ml; maximal stimulation was observed with 100-200 ng/ml. This effect was specific since THC did not affect gamma glutamyl transpeptidase activity or the secretion of plasminogen activator, lactate and transferrin. This observation that THC affects ABP secretion specifically is the first report of any differential effect of a drug on Sertoli cell secretion.

Androgen-Binding Protein↗